Assay for collagen degradation

ABSTRACT

The patent application describes a sandwich-type immunoassay for the detection and/or quantitation of collagen degradation products in biological samples such as blood, serum, plasma, sputum and cell cultures. The immunoassay uses a first antibody directed at an epitope present on a collagen molecule at a distance of up to 165 amino acids from a collagen telopeptide crosslink site, and a second antibody directed at another epitope of the crosslinked collagen molecule.

FIELD OF THE INVENTION

The invention relates to a method for detecting collagen degradation.More specifically, it relates to a method for quantitating highmolecular weight crosslinked collagen fragments in blood, plasma, serum,sputum or cell cultures.

BACKGROUND

Collagen proteins in connective tissues constitute a three-dimensionalnetwork with crosslinks. The crosslinks are formed after deposition ofthe collagen molecule in the extracellular matrix. Different types ofcrosslinks between collagen molecules are known. As a result fromenzymatic reactions, lysino-norleucine-type difunctional crosslinks,pyridinoline-type trifunctional crosslinks, and pyrrole-type crosslinksare formed. Aberrant degradation of collagen is an indication ofdisorders of connective tissues. Disorders of bone tissue compriseosteoporosis, Paget's disease, bone tumours, and drug-related bone loss.Disorders of cartilage tissue occur in arthritis (such asosteo-arthritis and rheumatoid arthritis), and disorders of thecardiovascular system are prominent in atherosclerosis, aneurisms,hypertension, myocardial infarction and hypertrophy. Similarly,disorders of the respiratory system and liver, such as fibrosis, of theteeth and periodontal tissues, of skin and wound healing, and in tumoursinvolve changes in collagen turnover.

Known methods to determine turnover of connective tissues, in particularbone, are based on serum/plasma/blood or urinary levels of pyridinolinecrosslinks (free or still containing residual amino acids), ortelopeptide sequences corresponding to the crosslinking site. Forexample U.S. Pat. No. 5,140,103 discloses a method for measuringcollagen degradation by quantitating the concentration of a C-terminalcollagen type II or III telopeptide containing a pyridinoline-typecrosslink by immunological, electrochemical or fluorometric assays.WO-92/21698 describes a method of determining bone resorption by meansof antibodies to type I collagen telopeptides. WO-95/08115 discloses amethod for assaying collagen fragments by means of antibodies againstcrosslinking sites of collagen. Similarly, WO-91/10141 describes the useof antibodies to detect collagen-derived crosslinks in biological fluidsas a diagnostic method of bone diseases.

A disadvantage of these known methods for assaying collagen degradationis that the detection means (e.g. antibodies) recognise an epitope thatincludes the crosslink, or an epitope close to the crosslinking site.Thus, these methods depend on the nature and structure of the crosslink,or the amino acid sequence close to the crosslinking site. Collagenfragments containing other types of crosslinks are not necessarilydetected and thus not quantitated, which leads to a less reliable and/orless sensitive result. More importantly, most of these methods require arelatively insensitive and laborious competition-type immunoassay thatis mostly only applicable to urine, and not to serum/plasma/blood.Disadvantages of urinary assays are the need to correct for the urinevolume based on creatinine levels, diurnal variation in urinaryexcretion rate and the need to collect urine samples.

STRUCTURE OF COLLAGEN CROSSLINKING

The formation of crosslinks in collagen is a basic feature of maturationof the tissue. The crosslinks can be intramolecular, linking twocollagen α-chains within the same molecule, or intermolecular, betweenchains in different molecules. Irrespective of the exact structure ofthe crosslink, by definition they involve two or more α-chains.

The chemistry and biology of crosslink formation have been reviewedextensively. The basic mechanism of crosslinking is oxidativedeamination of the α-amino groups of lysine or hydroxylysine residues,catalysed by lysyl oxidase. The lysine or hydroxylysine aldehydes formedundergo condensation reactions with lysine or hydroxylysine into thedifunctional crosslinks (dehydro)-lysinonorleucine,-hydroylysinonorleucine, and -dihydroxylysinonorleucine. Otherdifunctional crosslinks involve aldol condensation of two aldehydes, andpyrrole-type crosslinks.

Subsequently, trifunctional crosslinks connecting three α-chains mayresult: for instance, hydroxylysinonorleucine anddihydroxylysinonorleucine are converted into the pyridinoline crosslinkslysylpyridinoline (LP) and hydroxylysylpyridinoline (HP), respectively.The difunctional crosslink hydroxylysinonorleucine may also undergocondensation with histidinc to form the trifunctional crosslinkhistidinohydroxylysinonorleucine. Furthermore, the aldol product of twohydroxylysine aldehydes can undergo condensation with hydroxylysineresulting in hydroxymerodesmosine, or with histidine leading todehydroaldolhistidine. The latter can undergo additional condensationwith lysine or hydroxylysine to produce the tetrafunctional crosslinksdehydro-histidino-merodesmosine anddehydro-histidino-hydroxymerodesmosine, respectively.

The type of crosslink formation depends more on tissue localisation thanon the type of collagen. For instance, crosslinks based on lysinealdehydes predominate in skin. The crosslinks formed via thehydroxylysine pathway predominate in bone and cartilage. The di-, tri-or tetrafunctional crosslinks are reproducibly located at predefinedpositions in the collagen molecule, probably due to the selectivity oflysyl oxidase. For instance, difunctional hydroxylysinonorleucine anddihydroxylysinonorleucine and their trifunctional pyridinolinematuration products (LP and HP, respectively) in bone and cartilage(type I and II collagen, respectively) involve the lysine orhydroxylysine at position 87 in the triple helix and the hydroxylysineresidue in the C-terminal telopeptide. Furthermore, these crosslinks areformed between lysine or hydroxylysine at position 930 of the triplehelix and the hydroxylysine residue in the N-terminal telopeptide (seeFIG. 2).

In type II collagen, composed of three identical α₁ (II)-chains, bydefinition crosslinks are homotypic. Similarly, crosslinks of type IIIcollagen (composed of three α₁ (III)-chains) are homotypic. Homotypiccrosslinks in type I collagen may involve the α₁ (I)- as well as the α₂(I)-chain. Also heterotypic crosslinliks, connecting different collagenmolecules have been reported: crosslinks between type I and III collagenin blood vessels, between type II and IX, type II and IX, and type IIand III collagen in cartilage, between type I and V in bone, and betweentype I and II in intervertebral disc.

In summary, the enzymatic crosslinks are very diverse in nature, occurat specific locations in the molecule, and connect two or more collagenα-chains from the same or different types of collagen (homo- andheterotypic crosslinks).

SUMMARY OF THE INVENTION

It has been found according to the invention that collagen degradationcan be measured effectively in plasma, serum or blood by a sandwich-typeof immunoassay using epitopes further removed from the crosslinkingsite. The invention is concerned with a method of assaying collagendegradation products as defined in the appending claims; the method isgenerally applicable to crosslinked collagen fragments irrespective ofthe exact nature and structure of the crosslinking site. The inventionalso pertains to antibodies and diagnostic kits for carrying such amethod.

DETAILED DESCRIPTION OF THE INVENTION

It was found by HPLC analysis of normal serum that pyridinolinecrosslinks are present in collagen degradation products with relativelyhigh molecular weight (cf. Example 1; FIG. 1). About 25, 50 and 25% havemolecular weights between 0-10, 10-30, and 30-50 kD, respectively.Assuming an average weight per amino acid of 100 Dalton, half of thecollagen degradation products in serum contain between 100 and 300 aminoacids. One quarter of the fragments comprise 300-500 amino acids, whileanother quarter represents fragments with less than 100 amino acids.This implies that collagen degradation products present in blood can beas large as 500 amino acids. Thus, on average the three α-chains of thepyridinoline crosslink-containing degradation products are as large asup to 500/3=165 amino acids. Thus, these fragments contain not only(part of) the collagen telopeptide (telopeptides usually comprise about15 amino acids), but also large peptides, up to 165 amino acids, fromthe triple helical region (FIG. 2).

According to the invention high-molecular weight collagen fragments canbe detected by α-chain epitopes residing in the triple helical region,irrespective of the type of crosslink. Inasmuch as two, three or fourα-chains are present in these collagen degradation products in serum, asandwich-type of assay with one antibody recognising an epitope in oneα-chain, and a second antibody recognising another α-chain (which may bethe same type of α-chain), or even on the same α-chain as explainedbelow, is proposed. The two or more epitopes for the antibodies can bepresent on the degradation product at different locations as depicted inFIG. 3.

The epitopes suitable for the present method are located at a distanceof up to 165 amino acids from a collagen chain crosslink site. Adistance of an epitope to a crosslink site of a particular number ofamino acids is understood as that number of amino acids along the aminoacid chain from the distal amino acid of the epitope to the lysine orhydroxylysine residue of either a telopeptide crosslink site or a triplehelical region crosslink site, the latter being located at about 100amino acids distance from the collagen chain ends (see FIGS. 4 and 5).

An especially preferred distance from the crosslink sites (whethertelopeptide or triple helical) is up to 100 amino acids, especially5-100 or even 10-90 amino acids.

One of the epitopes should be present on a chain having a telopeptidecrosslink site. It may be present on the crosslink site itself and evencomprise part of the other chains linked by the crosslink. If the firstepitope is present on the telopeptide crosslink, the second epitope mayreside on the same chain, as depicted in FIG. 3, variations 3 and 5. Thesecond epitope may, however, also be present on another chain (FIG. 3,variations 2, 8 and 9). If the first epitope is at some distance(e.g. >5 amino acids) from the telopeptide crosslink, to the extent thatan antibody to the epitope does not discriminate for the presence of thecrosslink, the second epitope should be present on another α- chain, inorder to selectively detect degradation products that contain acrosslink (FIG. 3, variations 1, 4, 6 and 7). In this case, where thetwo α-chains are the same, the epitopes may be the same as well (FIG. 3,variation 7). A very suitable combination of epitopes (and antibodies)for the sandwich assay is a first epitope on a collagen within 165 aminoacids from a telopeptide crosslink, and a second epitope on anothercollagen chain within 165 amino acids from a triple helical regioncrosslink; this combination recognises most of the degradation productsthat are of diagnostic interest. If the telopeptide crosslink isN-terminal, the helical region crosslink is at the 930 or equivalentposition (FIG. 3, variations 1, 2, 4, 9), whereas if the telopeptidecrosslink is C-terminal, the helical region crosslink is at the 87 orequivalent position (not depicted in FIG. 3, but similarly applicable).

The antibodies according to the invention can be produced by well-knownmethods. Preferably the antibodies are monoclonal antibodies generatedby immunisation with either natural collagen degradation products havingmolecular weights of up to 50 kD, or with synthetic peptides containingat least the epitope of interest.

One of said antibodies is preferably immobilised in a known way, e.g. byphysical or covalent binding to spheres, beads, sticks, plate walls, andthe like, or immobilisable by aspecific reaction with a further,immobilised antibody.

Another of the other of said antibodies is preferably detectable bybeing provided with a detectable label, such as a fluorescence marker, aradioisotope, an enzyme, an avidine-biotine marker or the like, or bybeing recognised by a further, labelled antibody.

The invention further provides novel antibodies as described above,directed at an epitope which is at some distance, up to 165 amino acids,in particular 5-150 amino acids, more in particular 10 -100 amino acids,from a crosslink site. The invention also provides a diagnostic kitcontaining at least one of such antibodies, optionally two of saidantibodies, one being immobilised or immobilisable, the other beinglabelled or detectable. The kit may further contain conventional meansfor carrying out a sandwich-type immunoassay, such as enzyme substratesin case of enzyme labels, diluents, buffers, filters, etc.

DESCRIPTION OF THE FIGURES

FIG. 1. Molecular weight distribution, on a molar basis, of collagendegradation products containing pyridinoline crosslinks in normal humanserum.

FIG. 2. Schematic representation of collagen degradation productspresent in serum, based on findings presented in FIG. 1, and illustratedfor collagen types I, II and III. Fragments containing trifunctionalcrosslinks (e.g. pyridinolines) contain all 3 α-chains (A1, A2, and B;C1, C2, and D). Fragments with difunctional crosslinks are composed of 2α-chains (A2 and B; C2 and D). The maximal length of each peptide chainis on average 165 amino acids (α-chains B and D and A1, A2, C1 and C2some 15 thereof represent the N- or C-terminal telopeptide; theremaining α-chain of 150 (or less) amino acids α-chain are derived fromthe triple helical region). Italic numbers refer to residue numbers ofthe triple helix. Although the triple helix region fragments (B and Dare depicted with the crosslink site 930 or 87 in the middle, otherfragments are equally possible (e.g. 800-965 in B or and 35-200 in D, orshorter fragments).

FIG. 3. Principle of sandwich-type assay of high-molecular weightcrosslinked collagen degradation products in serum, plasma or blood,illustrated with degradation products composed of two or three α-chainscrosslinked at residue 930 of the triple helix and the N-terminaltelopeptide, originating from homotypically crosslinked collagens typeI, II or III. The antibody recognising the telopeptide (situations 2, 3,5, 8 and 9) may recognise any of the drawn telopeptides, as well asshared epitopes between two telopeptides, or telopeptide and triplehelical region (not depicted). Similar principles apply to the fragmentscontaining crosslinks between residue 87 and the C-terminal telopeptideof these collagens, as well as to heterotypically crosslinked fragmentsderived from combinations of collagens such as type I and II, I and III,I and V, II and III, II and IX, and II and XI.

One of the two antibodies drawn in each of the 9 possible situations(panel A) serves as a catching antibody. The other antibody, that may beidentical to the first, is used to detect the collagen degradationfragments, by whatever means, in a sandwich assay (see panel B).Antibodies directed against any epitope in situations 1 -9 may serve thepurpose of developing a sensitive and convenient assay to detect or/andquantitate collagen degradation products in serum, plasma or blood orother medium.

FIG. 4. Sequences of collagen types I, II and III proteins, withpossible sites of epitopes based on crosslinking residue in triplehelical region. The underlined parts represent epitopes based oncrosslinking residues in triple helical region; double underlined areresidues involved in lysyl oxidase-mediated crosslinking. P representsproline or hydroxyproline, K represents lysine or hydroxylysine(optionally glycosylated).

FIG. 5: Sequences of collagen types I, II and III proteins, withpossible sites of epitopes based on crosslinking residue intelopeptides. The underlined parts represent epitopes based oncrosslinking residues in telopeptides; double underlined are residuesinvolved in lysyl oxidase-mediated crosslinking. P represents proline orhydroxyproline, K represents lysine or hydroxylysine (optionallyglycosylated).

EXAMPLE 1

Normal human serum was separated into fractions with different molecularweight by ultrafiltration: serum (10 ml) was passed through cutoff-filters of 100, 50, 30, and 10 kD (Centriprep, Grace BV, AmiconDivision, Capelle aan de IJssel, The Netherlands. Fractions (0.5 ml)with molecular weight of <10 kD, 10-30 , 30-50 kD, and 50-100 kD weredried, reconstituted in 250 μl 6 N HCl, and hydrolysed at 108° C. for 16hours. Hydrolysed fractions were dried, reconstituted in 500 μl 6N HCl,200 μl glacial acetic acid, 200 μl CF-1 cellulose slurry, and 800 μln-butanol, and subjected to solid phase extraction with CF-1 celluloseaccording to Black et al, (Anal. Biochem. 1988, 169: 197-203).HPLChromatography was used for quantitating amounts of pyridinolinecrosslinks, which were detected by their fluoresence at 295/400 nm(excitation/emission wavelengths) (see Black et al. above).

Substantial amounts of pyridinoline crosslinks (the sum ofhydroxylysylpyridinoline and lysylpyridinoline) were detected in 0-10,10-30, and 30-50 kD fractions. About half of the total serumconcentration of pyridinoline (approx. 1.5 nM) had a molecular weightbetween 10 and 30 kD. About 25% had a molecular weight less than 10 kD.The other 25% had molecular weight between 30 and 50 kD (FIG. 1).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1057 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                                (F) TISSUE TYPE: Collag - #en type I                                #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Th - #r Gly Gly Ile Ser Val         #                15                                                           - Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gl - #y Leu Pro Gly Pro Pro         #            30                                                               - Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pr - #o Pro Gly Glu Pro Gly         #        45                                                                   - Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Ar - #g Gly Pro Pro Gly Pro         #    60                                                                       - Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gl - #y Lys Pro Gly Arg Pro         #80                                                                           - Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Al - #a Arg Gly Leu Pro Gly         #                95                                                           - Thr Ala Gly Leu Pro Gly Met Lys Gly His Ar - #g Gly Phe Ser Gly Leu         #           110                                                               - Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gl - #y Pro Lys Gly Glu Pro         #       125                                                                   - Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gl - #n Met Gly Pro Arg Gly         #   140                                                                       - Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pr - #o Gly Pro Ala Gly Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gl - #y Pro Pro Gly Pro Thr         #               175                                                           - Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Al - #a Val Gly Ala Lys Gly         #           190                                                               - Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Gl - #u Gly Pro Gln Gly Val         #       205                                                                   - Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gl - #y Ala Ala Gly Pro Ala         #   220                                                                       - Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Al - #a Lys Gly Ala Asn Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pr - #o Gly Ala Arg Gly Pro         #               255                                                           - Ser Gly Pro Gln Gly Pro Gly Gly Pro Thr Gl - #y Ala Arg Gly Leu Val         #           270                                                               - Gly Glu Pro Gly Pro Ala Gly Ser Lys Gly Gl - #u Ser Gly Asn Lys Gly         #       285                                                                   - Glu Pro Gly Ser Ala Gly Pro Gln Gly Pro Pr - #o Gly Pro Ser Gly Glu         #   300                                                                       - Glu Gly Lys Arg Gly Pro Asn Gly Glu Ala Gl - #y Ser Ala Gly Pro Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gly Pro Pro Gly Leu Arg Gly Gly Pro Gly Se - #r Arg Gly Phe Pro Gly         #               335                                                           - Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Al - #a Gly Glu Arg Gly Ser         #           350                                                               - Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gl - #y Glu Ala Gly Arg Pro         #       365                                                                   - Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Le - #u Thr Gly Ser Pro Gly         #   380                                                                       - Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pr - #o Gly Pro Ala Gly Gln         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gl - #y Ala Arg Gly Gln Ala         #               415                                                           - Gly Val Met Gly Phe Pro Gly Pro Lys Gly Al - #a Ala Gly Glu Pro Gly         #           430                                                               - Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pr - #o Gly Ala Val Gly Pro         #       445                                                                   - Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gl - #y Pro Pro Gly Pro Ala         #   460                                                                       - Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pr - #o Ala Gly Ser Pro Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pr - #o Gly Glu Ala Gly Lys         #               495                                                           - Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gl - #y Ala Pro Gly Pro Ser         #           510                                                               - Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Gl - #u Arg Gly Val Gln Gly         #       525                                                                   - Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala As - #n Gly Ala Pro Gly Asn         #   540                                                                       - Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gl - #y Ala Pro Gly Ser Gln         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Gl - #u Arg Gly Ala Ala Gly         #               575                                                           - Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Al - #a Gly Pro Lys Gly Ala         #           590                                                               - Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gl - #y Leu Thr Gly Pro Ile         #       605                                                                   - Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly As - #p Lys Gly Glu Ser Gly         #   620                                                                       - Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Ar - #g Gly Ala Pro Gly Asp         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gl - #y Phe Ala Gly Pro Pro         #               655                                                           - Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Gl - #u Pro Gly Asp Ala Gly         #           670                                                               - Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Al - #a Gly Pro Ala Gly Pro         #       685                                                                   - Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gl - #y Ala Lys Gly Ala Arg         #   700                                                                       - Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Ph - #e Pro Gly Ala Ala Gly         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Al - #a Gly Pro Pro Gly Pro         #               735                                                           - Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gl - #y Pro Arg Gly Glu Thr         #           750                                                               - Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pr - #o Pro Gly Pro Pro Gly         #       765                                                                   - Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala As - #p Gly Pro Ala Gly Ala         #   780                                                                       - Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gl - #y Gln Arg Gly Val Val         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Ph - #e Pro Gly Leu Pro Gly         #               815                                                           - Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Se - #r Gly Ala Ser Gly Glu         #           830                                                               - Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gl - #y Leu Ala Gly Pro Pro         #       845                                                                   - Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Al - #a Glu Gly Ser Pro Gly         #   860                                                                       - Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Ar - #g Gly Glu Thr Gly Pro         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gl - #y Ala Pro Gly Pro Val         #               895                                                           - Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Gl - #u Thr Gly Pro Ala Gly         #           910                                                               - Pro Ala Gly Pro Val Gly Pro Ala Gly Ala Ar - #g Gly Pro Ala Gly Pro         #       925                                                                   - Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gl - #y Glu Gln Gly Asp Arg         #   940                                                                       - Gly Ile Lys Gly His Arg Gly Phe Ser Gly Le - #u Gln Gly Pro Pro Gly         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Se - #r Gly Ala Ser Gly Pro         #               975                                                           - Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gl - #y Ala Pro Gly Lys Asp         #           990                                                               - Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pr - #o Pro Gly Pro Arg Gly         #      10050                                                                  - Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pr - #o Gly Pro Pro Gly Pro         #  10205                                                                      - Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Ph - #e Asp Phe Ser Phe Leu         #               10401030 - #                1035                              - Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gl - #y Gly Arg Tyr Tyr Arg         #              10550                                                          - Ala                                                                         - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1024 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                                (F) TISSUE TYPE: Collag - #en type I                                #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Gln Tyr Asp Gly Lys Gly Val Gly Leu Gly Pr - #o Gly Pro Met Gly Leu         #                15                                                           - Met Gly Pro Arg Gly Pro Pro Gly Ala Ala Gl - #y Ala Pro Gly Pro Gln         #            30                                                               - Gly Phe Gln Gly Pro Ala Gly Glu Pro Gly Gl - #u Pro Gly Gln Thr Gly         #        45                                                                   - Pro Ala Gly Ala Arg Gly Pro Ala Gly Pro Pr - #o Gly Lys Ala Gly Glu         #    60                                                                       - Asp Gly His Pro Gly Lys Pro Gly Arg Pro Gl - #y Glu Arg Gly Val Val         #80                                                                           - Gly Pro Gln Gly Ala Arg Gly Phe Pro Gly Th - #r Pro Gly Leu Pro Gly         #                95                                                           - Phe Lys Gly Ile Arg Gly His Asn Gly Leu As - #p Gly Leu Lys Gly Gln         #           110                                                               - Pro Gly Ala Pro Gly Val Lys Gly Glu Pro Gl - #y Ala Pro Gly Glu Asn         #       125                                                                   - Gly Thr Pro Gly Gln Thr Gly Ala Arg Gly Le - #u Pro Gly Glu Arg Gly         #   140                                                                       - Arg Val Gly Ala Pro Gly Pro Ala Gly Ala Ar - #g Gly Ser Asp Gly Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Gly Pro Val Gly Pro Ala Gly Pro Asn Gl - #y Ser Ala Gly Pro Pro         #               175                                                           - Gly Phe Pro Gly Ala Pro Gly Pro Lys Gly Gl - #u Ile Gly Ala Val Gly         #           190                                                               - Asn Ala Gly Pro Thr Gly Pro Ala Gly Pro Ar - #g Gly Glu Val Gly Leu         #       205                                                                   - Pro Gly Leu Ser Gly Pro Val Gly Pro Pro Gl - #y Asn Pro Gly Ala Asn         #   220                                                                       - Gly Leu Thr Gly Ala Lys Gly Ala Ala Gly Le - #u Pro Gly Val Ala Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Pro Gly Leu Pro Gly Pro Arg Gly Ile Pr - #o Gly Pro Pro Gly Ala         #               255                                                           - Ala Gly Thr Thr Gly Ala Arg Gly Leu Val Gl - #y Glu Pro Gly Pro Ala         #           270                                                               - Gly Ser Lys Gly Glu Ser Gly Asn Lys Gly Gl - #u Pro Gly Ser Ala Gly         #       285                                                                   - Pro Gln Gly Pro Pro Gly Pro Ser Gly Glu Gl - #u Gly Lys Arg Gly Pro         #   300                                                                       - Asn Gly Glu Ala Gly Ser Ala Gly Pro Pro Gl - #y Pro Pro Gly Leu Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gly Ser Pro Gly Ser Arg Gly Leu Pro Gly Al - #a Asp Gly Arg Ala Gly         #               335                                                           - Val Met Gly Pro Pro Gly Ser Arg Gly Ala Se - #r Gly Pro Ala Gly Val         #           350                                                               - Arg Gly Pro Asn Gly Asp Ala Gly Arg Pro Gl - #y Glu Pro Gly Leu Met         #       365                                                                   - Gly Pro Arg Gly Leu Pro Gly Ser Pro Gly As - #n Ile Gly Pro Ala Gly         #   380                                                                       - Lys Glu Gly Pro Val Gly Leu Pro Gly Ile As - #p Gly Arg Pro Gly Pro         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ile Gly Pro Val Gly Ala Arg Gly Glu Pro Gl - #y Asn Ile Gly Phe Pro         #               415                                                           - Gly Pro Lys Gly Pro Thr Gly Asp Pro Gly Ly - #s Asn Gly Asp Lys Gly         #           430                                                               - His Ala Gly Leu Ala Gly Ala Arg Gly Ala Pr - #o Gly Pro Asp Gly Asn         #       445                                                                   - Asn Gly Ala Gln Gly Pro Pro Gly Pro Gln Gl - #y Val Gln Gly Gly Lys         #   460                                                                       - Gly Glu Gln Gly Pro Ala Gly Pro Pro Gly Ph - #e Gln Gly Leu Pro Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Pro Ser Gly Pro Ala Gly Glu Val Gly Lys Pr - #o Gly Glu Arg Gly Leu         #               495                                                           - His Gly Glu Phe Gly Leu Pro Gly Pro Ala Gl - #y Pro Arg Gly Glu Arg         #           510                                                               - Gly Pro Pro Gly Glu Ser Gly Ala Ala Gly Pr - #o Thr Gly Pro Ile Gly         #       525                                                                   - Ser Arg Gly Pro Ser Gly Pro Pro Gly Pro As - #p Gly Asn Lys Gly Glu         #   540                                                                       - Pro Gly Val Val Gly Ala Val Gly Thr Ala Gl - #y Pro Ser Gly Pro Ser         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Gly Leu Pro Gly Glu Arg Gly Ala Ala Gly Il - #e Pro Gly Gly Lys Gly         #               575                                                           - Glu Lys Gly Glu Pro Gly Leu Arg Gly Glu Il - #e Gly Asn Pro Gly Arg         #           590                                                               - Asp Gly Ala Arg Gly Ala His Gly Ala Val Gl - #y Ala Pro Gly Pro Ala         #       605                                                                   - Gly Ala Thr Gly Asp Arg Gly Glu Ala Gly Al - #a Ala Gly Pro Ala Gly         #   620                                                                       - Pro Ala Gly Pro Arg Gly Ser Pro Gly Glu Ar - #g Gly Glu Val Gly Pro         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ala Gly Pro Asn Gly Phe Ala Gly Pro Ala Gl - #y Ala Ala Gly Gln Pro         #               655                                                           - Gly Ala Lys Gly Glu Arg Gly Gly Lys Gly Pr - #o Lys Gly Glu Asn Gly         #           670                                                               - Val Val Gly Pro Thr Gly Pro Val Gly Ala Al - #a Gly Pro Ala Gly Pro         #       685                                                                   - Asn Gly Pro Pro Gly Pro Ala Gly Ser Arg Gl - #y Asp Gly Gly Pro Pro         #   700                                                                       - Gly Met Thr Gly Phe Pro Gly Ala Ala Gly Ar - #g Thr Gly Pro Pro Gly         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Pro Ser Gly Ile Ser Gly Pro Pro Gly Pro Pr - #o Gly Pro Ala Gly Lys         #               735                                                           - Glu Gly Leu Arg Gly Pro Arg Gly Asp Gln Gl - #y Pro Val Gly Arg Thr         #           750                                                               - Gly Glu Val Gly Ala Val Gly Pro Pro Gly Ph - #e Ala Gly Glu Lys Gly         #       765                                                                   - Pro Ser Gly Glu Ala Gly Thr Ala Gly Pro Pr - #o Gly Thr Pro Gly Pro         #   780                                                                       - Gln Gly Leu Leu Gly Ala Pro Gly Ile Leu Gl - #y Leu Pro Gly Ser Arg         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Gly Glu Arg Gly Leu Pro Gly Val Ala Gly Al - #a Val Gly Glu Pro Gly         #               815                                                           - Pro Leu Gly Ile Ala Gly Pro Pro Gly Ala Ar - #g Gly Pro Pro Gly Ala         #           830                                                               - Val Gly Ser Pro Gly Val Asn Gly Ala Pro Gl - #y Glu Ala Gly Arg Asp         #       845                                                                   - Gly Asn Pro Gly Asn Asp Gly Pro Pro Gly Ar - #g Asp Gly Gln Pro Gly         #   860                                                                       - His Lys Gly Glu Arg Gly Tyr Pro Gly Asn Il - #e Gly Pro Val Gly Ala         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Ala Gly Ala Pro Gly Pro His Gly Pro Val Gl - #y Pro Ala Gly Lys His         #               895                                                           - Gly Asn Arg Gly Glu Thr Gly Pro Ser Gly Pr - #o Val Gly Pro Ala Gly         #           910                                                               - Ala Val Gly Pro Arg Gly Pro Ser Gly Pro Gl - #n Gly Ile Arg Gly Asp         #       925                                                                   - Lys Gly Glu Pro Gly Glu Lys Gly Pro Arg Gl - #y Leu Pro Gly Phe Lys         #   940                                                                       - Gly His Asn Gly Leu Gln Gly Leu Pro Gly Il - #e Ala Gly His His Gly         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Asp Gln Gly Ala Pro Gly Ser Val Gly Pro Al - #a Gly Pro Arg Gly Pro         #               975                                                           - Ala Gly Pro Ser Gly Pro Ala Gly Lys Asp Gl - #y Arg Thr Gly His Pro         #           990                                                               - Gly Thr Val Gly Pro Ala Gly Ile Arg Gly Pr - #o Gln Gly His Gln Gly         #      10050                                                                  - Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro Le - #u Gly Pro Leu Gly Val         #  10205                                                                      - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1060 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                                (F) TISSUE TYPE: Collag - #en type II                               #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Gln Met Ala Gly Gly Phe Asp Glu Lys Ala Gl - #y Gly Ala Gln Leu Gly         #                15                                                           - Val Met Gln Gly Pro Met Gly Pro Met Gly Pr - #o Arg Gly Pro Pro Gly         #            30                                                               - Pro Ala Gly Ala Pro Gly Pro Gln Gly Phe Gl - #n Gly Asn Pro Gly Glu         #        45                                                                   - Pro Gly Glu Pro Gly Val Ser Gly Pro Met Gl - #y Pro Arg Gly Pro Pro         #    60                                                                       - Gly Pro Pro Gly Lys Pro Gly Asp Asp Gly Gl - #u Ala Gly Lys Pro Gly         #80                                                                           - Lys Ala Gly Glu Arg Gly Pro Pro Gly Pro Gl - #n Gly Ala Arg Gly Phe         #                95                                                           - Pro Gly Thr Pro Gly Leu Pro Gly Val Lys Gl - #y His Arg Gly Tyr Pro         #           110                                                               - Gly Leu Asp Gly Ala Lys Gly Glu Ala Gly Al - #a Pro Gly Val Lys Gly         #       125                                                                   - Glu Ser Gly Ser Pro Gly Glu Asn Gly Ser Pr - #o Gly Pro Met Gly Pro         #   140                                                                       - Arg Gly Leu Pro Gly Glu Arg Gly Arg Thr Gl - #y Pro Ala Gly Ala Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Ala Arg Gly Asn Asp Gly Gln Pro Gly Pr - #o Ala Gly Pro Pro Gly         #               175                                                           - Pro Val Gly Pro Ala Gly Gly Pro Gly Phe Pr - #o Gly Ala Pro Gly Ala         #           190                                                               - Lys Gly Glu Ala Gly Pro Thr Gly Ala Arg Gl - #y Pro Glu Gly Ala Gln         #       205                                                                   - Gly Pro Arg Gly Glu Pro Gly Thr Pro Gly Se - #r Pro Gly Pro Ala Gly         #   220                                                                       - Ala Ser Gly Asn Pro Gly Thr Asp Gly Ile Pr - #o Gly Ala Lys Gly Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Gly Ala Pro Gly Ile Ala Gly Ala Pro Gl - #y Phe Pro Gly Pro Arg         #               255                                                           - Gly Pro Pro Asp Pro Gln Gly Ala Thr Gly Pr - #o Leu Gly Pro Lys Gly         #           270                                                               - Gln Thr Gly Lys Pro Gly Ile Ala Gly Phe Ly - #s Gly Glu Gln Gly Pro         #       285                                                                   - Lys Gly Glu Pro Gly Pro Ala Gly Pro Gln Gl - #y Ala Pro Gly Pro Ala         #   300                                                                       - Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly Gl - #u Pro Gly Gly Val Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Ile Gly Pro Pro Gly Glu Arg Gly Ala Pr - #o Gly Asn Arg Gly Phe         #               335                                                           - Pro Gly Gln Asp Gly Leu Ala Gly Pro Lys Gl - #y Ala Pro Gly Glu Arg         #           350                                                               - Gly Pro Ser Gly Leu Ala Gly Pro Lys Gly Al - #a Asn Gly Asp Pro Gly         #       365                                                                   - Arg Pro Gly Glu Pro Gly Leu Pro Gly Ala Ar - #g Gly Leu Thr Gly Arg         #   380                                                                       - Pro Gly Asp Ala Gly Pro Gln Gly Lys Val Gl - #y Pro Ser Gly Ala Pro         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Glu Asp Gly Arg Pro Gly Pro Pro Gly Pr - #o Gln Gly Ala Arg Gly         #               415                                                           - Gln Pro Gly Val Met Gly Phe Pro Gly Pro Ly - #s Gly Ala Asn Gly Glu         #           430                                                               - Pro Gly Lys Ala Gly Glu Lys Gly Leu Pro Gl - #y Ala Pro Gly Leu Arg         #       445                                                                   - Gly Leu Pro Gly Lys Asp Gly Glu Thr Gly Al - #a Glu Gly Pro Pro Gly         #   460                                                                       - Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu Gl - #n Gly Ala Pro Gly Pro         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ser Gly Phe Gln Gly Leu Pro Gly Pro Pro Gl - #y Pro Pro Gly Glu Gly         #               495                                                           - Gly Lys Pro Gly Asp Gln Gly Val Pro Gly Gl - #u Ala Gly Ala Pro Gly         #           510                                                               - Leu Val Gly Pro Arg Gly Glu Arg Gly Phe Pr - #o Gly Glu Arg Gly Ser         #       525                                                                   - Pro Gly Ala Gln Gly Leu Gln Gly Pro Arg Gl - #y Leu Pro Gly Thr Pro         #   540                                                                       - Gly Thr Asp Gly Pro Lys Gly Ala Ser Gly Pr - #o Ala Gly Pro Pro Gly         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ala Gln Gly Pro Pro Gly Leu Gln Gly Met Pr - #o Gly Glu Arg Gly Ala         #               575                                                           - Ala Gly Ile Ala Gly Pro Lys Gly Asp Arg Gl - #y Asp Val Gly Glu Lys         #           590                                                               - Gly Pro Glu Gly Ala Pro Gly Lys Asp Gly Gl - #y Arg Gly Leu Thr Gly         #       605                                                                   - Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala As - #n Gly Glu Lys Gly Glu         #   620                                                                       - Val Gly Pro Pro Gly Pro Ala Gly Ser Ala Gl - #y Ala Arg Gly Ala Pro         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Gly Glu Arg Gly Glu Thr Gly Pro Pro Gly Th - #r Ser Gly Ile Ala Gly         #               655                                                           - Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala Ly - #s Gly Glu Gln Gly Glu         #           670                                                               - Ala Gly Gln Lys Gly Asp Ala Gly Ala Pro Gl - #y Pro Gln Gly Pro Ser         #       685                                                                   - Gly Ala Pro Gly Pro Gln Gly Pro Thr Gly Va - #l Thr Gly Pro Lys Gly         #   700                                                                       - Ala Arg Gly Ala Gln Gly Pro Pro Gly Ala Th - #r Gly Phe Pro Gly Ala         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Ala Gly Arg Val Gly Pro Pro Gly Ser Asn Gl - #y Asn Pro Gly Pro Pro         #               735                                                           - Gly Pro Pro Gly Pro Ser Gly Lys Asp Gly Pr - #o Lys Gly Ala Arg Gly         #           750                                                               - Asp Ser Gly Pro Pro Gly Arg Ala Gly Glu Pr - #o Gly Leu Gln Gly Pro         #       765                                                                   - Ala Gly Pro Pro Gly Glu Lys Gly Glu Pro Gl - #y Asp Asp Gly Pro Ser         #   780                                                                       - Gly Ala Glu Gly Pro Pro Gly Pro Gln Gly Le - #u Ala Gly Gln Arg Gly         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Ar - #g Gly Phe Pro Gly Leu         #               815                                                           - Pro Gly Pro Ser Gly Glu Pro Gly Gln Gln Gl - #y Ala Pro Gly Ala Ser         #           830                                                               - Gly Asp Arg Gly Pro Pro Gly Pro Val Gly Pr - #o Pro Gly Leu Thr Gly         #       845                                                                   - Pro Ala Gly Glu Pro Gly Arg Glu Gly Ser Pr - #o Gly Ala Asp Gly Pro         #   860                                                                       - Pro Gly Arg Asp Gly Ala Ala Gly Val Lys Gl - #y Asp Arg Gly Glu Thr         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Gly Ala Val Gly Ala Pro Gly Ala Pro Gly Pr - #o Pro Gly Ser Pro Gly         #               895                                                           - Pro Ala Gly Pro Thr Gly Lys Gln Gly Asp Ar - #g Gly Glu Ala Gly Ala         #           910                                                               - Gln Gly Pro Met Gly Pro Ser Gly Pro Ala Gl - #y Ala Arg Gly Ile Gln         #       925                                                                   - Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Gl - #u Ala Gly Glu Pro Gly         #   940                                                                       - Glu Arg Gly Leu Lys Gly His Arg Gly Phe Th - #r Gly Leu Gln Gly Leu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Pro Gly Pro Pro Gly Pro Ser Gly Asp Gln Gl - #y Ala Ser Gly Pro Ala         #               975                                                           - Gly Pro Ser Gly Pro Arg Gly Pro Pro Gly Pr - #o Val Gly Pro Ser Gly         #           990                                                               - Lys Asp Gly Ala Asn Gly Ile Pro Gly Pro Il - #e Gly Pro Pro Gly Pro         #      10050                                                                  - Arg Gly Arg Ser Gly Glu Thr Gly Pro Ala Gl - #y Pro Pro Gly Asn Pro         #  10205                                                                      - Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pr - #o Gly Ile Asp Met Ser         #               10401030 - #                1035                              - Ala Phe Ala Gly Leu Gly Pro Arg Glu Lys Gl - #y Pro Asp Pro Leu Gln         #              10550                                                          - Tyr Met Arg Ala                                                                         1060                                                              - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1057 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                                (F) TISSUE TYPE: Collag - #en type III                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1055                                                  #/label= ModifiedER INFORMATION:                                              #"Ala may be Pro"ote=                                                         #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Gln Asn Tyr Ser Pro Gln Tyr Asp Ser Tyr As - #p Val Lys Ser Gly Val         #                15                                                           - Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pr - #o Ala Gly Pro Pro Gly         #            30                                                               - Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pr - #o Gly Ser Pro Gly Ser         #        45                                                                   - Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gl - #y Gln Ala Gly Pro Ser         #    60                                                                       - Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pr - #o Ser Gly Pro Ala Gly         #80                                                                           - Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pr - #o Gly Glu Arg Gly Leu         #                95                                                           - Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gl - #y Ile Pro Gly Phe Pro         #           110                                                               - Gly Met Lys Gly His Arg Gly Phe Asp Gly Ar - #g Asn Gly Glu Lys Gly         #       125                                                                   - Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu As - #n Gly Leu Pro Gly Glu         #   140                                                                       - Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gl - #y Ala Pro Gly Glu Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Al - #a Arg Gly Asn Asp Gly         #               175                                                           - Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pr - #o Gly Pro Pro Gly Thr         #           190                                                               - Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gl - #y Glu Val Gly Pro Ala         #       205                                                                   - Gly Ser Pro Gly Ser Asn Gly Ala Pro Gly Gl - #n Arg Gly Glu Pro Gly         #   220                                                                       - Pro Gln Gly His Ala Gly Ala Gln Gly Pro Pr - #o Gly Pro Pro Gly Ile         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Gly Ser Pro Gly Gly Lys Gly Glu Met Gl - #y Pro Ala Gly Ile Pro         #               255                                                           - Gly Ala Pro Gly Leu Met Gly Ala Arg Gly Pr - #o Pro Gly Pro Ala Gly         #           270                                                               - Ala Asn Gly Ala Pro Gly Leu Arg Gly Gly Al - #a Gly Glu Pro Gly Lys         #       285                                                                   - Asn Gly Ala Lys Gly Glu Pro Gly Pro Arg Gl - #y Glu Arg Gly Glu Ala         #   300                                                                       - Gly Ile Pro Gly Val Pro Gly Ala Lys Gly Gl - #u Asp Gly Lys Asp Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Pro Gly Glu Pro Gly Ala Asn Gly Leu Pr - #o Gly Ala Ala Gly Glu         #               335                                                           - Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gl - #y Pro Asn Gly Ile Pro         #           350                                                               - Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Al - #a Pro Gly Pro Ala Gly         #       365                                                                   - Pro Arg Gly Ala Ala Gly Glu Pro Gly Arg As - #p Gly Val Pro Gly Gly         #   380                                                                       - Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gl - #y Gly Pro Gly Ser Asp         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Gl - #u Ser Gly Arg Pro Gly         #               415                                                           - Pro Pro Gly Pro Ser Gly Pro Arg Gly Gln Pr - #o Gly Val Met Gly Phe         #           430                                                               - Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro Gl - #y Lys Asn Gly Glu Arg         #       445                                                                   - Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pr - #o Pro Gly Lys Asn Gly         #   460                                                                       - Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Th - #r Gly Pro Gly Gly Asp         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Lys Gly Asp Thr Gly Pro Pro Gly Pro Gln Gl - #y Leu Gln Gly Leu Pro         #               495                                                           - Gly Thr Gly Gly Pro Pro Gly Glu Asn Gly Ly - #s Pro Gly Glu Pro Gly         #           510                                                               - Pro Lys Gly Asp Ala Gly Ala Pro Gly Ala Pr - #o Gly Gly Lys Gly Asp         #       525                                                                   - Ala Gly Ala Pro Gly Glu Arg Gly Pro Pro Gl - #y Leu Ala Gly Ala Pro         #   540                                                                       - Gly Leu Arg Gly Gly Ala Gly Pro Pro Gly Pr - #o Glu Gly Gly Lys Gly         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ala Ala Gly Pro Pro Gly Pro Pro Gly Ala Al - #a Gly Thr Pro Gly Leu         #               575                                                           - Gln Gly Met Pro Gly Glu Arg Gly Gly Leu Gl - #y Ser Pro Gly Pro Lys         #           590                                                               - Gly Asp Lys Gly Glu Pro Gly Gly Pro Gly Al - #a Asp Gly Val Pro Gly         #       605                                                                   - Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Il - #e Gly Pro Pro Gly Pro         #   620                                                                       - Ala Gly Gln Pro Gly Asp Lys Gly Glu Gly Gl - #y Ala Pro Gly Leu Pro         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Gly Ile Ala Gly Pro Arg Gly Ser Pro Gly Gl - #u Arg Gly Glu Thr Gly         #               655                                                           - Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pr - #o Gly Gln Asn Gly Glu         #           670                                                               - Pro Gly Gly Lys Gly Glu Arg Gly Ala Pro Gl - #y Glu Lys Gly Glu Gly         #       685                                                                   - Gly Pro Pro Gly Val Ala Gly Pro Pro Gly Gl - #y Ser Gly Pro Ala Gly         #   700                                                                       - Pro Pro Gly Pro Gln Gly Val Lys Gly Glu Ar - #g Gly Ser Pro Gly Gly         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Pro Gly Ala Ala Gly Phe Pro Gly Ala Arg Gl - #y Leu Pro Gly Pro Pro         #               735                                                           - Gly Ser Asn Gly Asn Pro Gly Pro Pro Gly Pr - #o Ser Gly Ser Pro Gly         #           750                                                               - Lys Asp Gly Pro Pro Gly Pro Ala Gly Asn Th - #r Gly Ala Pro Gly Ser         #       765                                                                   - Pro Gly Val Ser Gly Pro Lys Gly Asp Ala Gl - #y Gln Pro Gly Glu Lys         #   780                                                                       - Gly Ser Pro Gly Ala Gln Gly Pro Pro Gly Al - #a Pro Gly Pro Leu Gly         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Ile Ala Gly Ile Thr Gly Ala Arg Gly Leu Al - #a Gly Pro Pro Gly Met         #               815                                                           - Pro Gly Pro Arg Gly Ser Pro Gly Pro Gln Gl - #y Val Lys Gly Glu Ser         #           830                                                               - Gly Lys Pro Gly Ala Asn Gly Leu Ser Gly Gl - #u Arg Gly Pro Pro Gly         #       845                                                                   - Pro Gln Gly Leu Pro Gly Leu Ala Gly Thr Al - #a Gly Glu Pro Gly Arg         #   860                                                                       - Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gl - #y Arg Asp Gly Ser Pro         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Gly Gly Lys Gly Asp Arg Gly Glu Asn Gly Se - #r Pro Gly Ala Pro Gly         #               895                                                           - Ala Pro Gly His Pro Gly Pro Pro Gly Pro Va - #l Gly Pro Ala Gly Lys         #           910                                                               - Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala Gl - #y Pro Ala Gly Ala Pro         #       925                                                                   - Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Pr - #o Gln Gly Pro Arg Gly         #   940                                                                       - Asp Lys Gly Glu Thr Gly Glu Arg Gly Ala Al - #a Gly Ile Lys Gly His         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gl - #y Ser Pro Gly Pro Ala         #               975                                                           - Gly Gln Gln Gly Ala Ile Gly Ser Pro Gly Pr - #o Ala Gly Pro Arg Gly         #           990                                                               - Pro Val Gly Pro Ser Gly Pro Pro Gly Lys As - #p Gly Thr Ser Gly His         #      10050                                                                  - Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gl - #y Asn Arg Gly Glu Arg         #  10205                                                                      - Gly Ser Glu Gly Ser Pro Gly His Pro Gly Gl - #n Pro Gly Pro Pro Gly         #               10401030 - #                1035                              - Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gl - #y Val Gly Ala Ala Ala         #              10550                                                          - Ile                                                                         __________________________________________________________________________

What is claim is:
 1. Method for assaying collagen degradation byassaying degradation products containing a collagen crosslink in abiological sample other than urine, which comprises (a) contacting saidsample, or a fraction thereof containing protein material, with a firstantibody directed at a first epitope present on a crosslinked collagenmolecule, said first epitope being located at a distance of up to 165amino acids from a collagen telopeptide crosslink site, and a secondantibody directed at a second epitope present on said collagen molecule,said second epitope being located at a distance of up to 165 amino acidsfrom a collagen crosslink site, at least one of said first and secondepitopes being located at a distance of at least 5 amino acids from acollagen crosslink site, and (b) detecting a complex formed by saidcollagen degradation product and said first and said second antibody. 2.Method according to claim 1, wherein one of said antibodies isdetectable and the other of said antibodies is immobilised.
 3. Methodaccording to claim 1, wherein said second epitope is present on acollagen molecule at a distance of 5 to 150 amino acids from a collagentriple helical region crosslink site.
 4. Method according to claim 1,wherein said second epitope is present on a collagen molecule at adistance of 5 to 165 amino acids from a collagen telopeptide crosslinksite.
 5. Method according to claim 2, for assaying degradation ofcollagen having at least two identical α-chains, wherein said firstantibody and said second antibody are directed at an identical epitopepresent on said identical α-chains of a collagen molecule at a distanceof from 5 to 165 amino acids from a collagen telopeptide crosslink site.6. Method according to claim 1, wherein said first epitope is present ona collagen telopeptide crosslink site, and said second epitope ispresent on a collagen molecule at a distance of 5 to 165 amino acidsfrom a collagen crosslink site.
 7. Method according to claim 1, whereinat least one of said first and said second epitope is located at adistance of 5 to 100 amino acids from said collagen crosslink site. 8.Method according to claim 1, wherein at least one of said first and saidsecond epitope is present on the collagen α1(I), α2(I), α1(II) orα1(III) chain or an α-chain of collagen type IV, V, VI, IX, X or XI. 9.Method according to claim 1, wherein said protein material has molecularweights between 3 kD and 50 kD, in particular between 5 kD and 40 kD.10. Method according claim 1, wherein said biological sample is derivedfrom blood, serum, plasma or sputum.
 11. Diagnostic kit for assayingbone degradation products containing a collagen crosslink, the kitcontaining at least first and second antibodies directed at first andsecond epitopes, respectively, present on a crosslinked collagenmolecule, at least one of said antibodies being directed at an epitopelocated at a distance of up to 165 amino acids from a collagentelopeptide crosslink site, of said collagen molecule, and at least oneof said antibodies being directed at an epitope located at a distance of5 to 165 amino acids from a collagen crosslink site of said collagenmolecule.